Somatic (adult) stem cells maintain tissue homeostasis by exhibiting long-term replicative potential, together with the capacities of self-renewal and multi-lineage differentiation. These stem cell properties are tightly regulated in normal development and their alteration can lead to tumorigenesis and metastases (Martínez-Climent et al., “Somatic Stem Cells and the Origin of Cancer,” Clin Transl Oncol. 8(9):647-63 (2006); Deleyrolle et al., “Determination of Somatic and Cancer Stem Cell Self-renewing Symmetric Division Rate Using Sphere Assays,” PLoS One 6(1):e15844 (2011)).
Cancer metastases occurs when cells leave the primary tumor, enter circulation, extravasate to a secondary site to initiate a new tumor (Fidler, “The Pathogenesis of Cancer Metastasis: The ‘Seed and Soil’ Hypothesis Revisited,” Nat Rev Cancer 3(6):453e8 (2003)). It is estimated that approximately one million cells are shed per gram of a tumor every day (Chang et al., “Mosaic Blood Vessels in Tumors: Frequency of Cancer Cells in Contact with Flowing Blood,” Proc Natl Acad Sci USA 97(26):14608e13 (2000)). However, only a few of these shed cells have the ability to metastasize (Zhou et al., “Tumour Initiating Cells: Challenges and Opportunities for Anticancer Drug Discovery,” Nat Rev Drug Discov 8(10):806e23 (2009); Held et al., “Characterization of Melanoma Cells Capable of Propagating Tumors from a Single Cell,” Cancer Res 70(1):388e97 (2010)). For a self-reliant circulating tumor cell to successfully metastasize it must possess the ability to interact with and condition the local microenvironment (Ireland et al., “Genetic Factors in Metastatic Progression of Cutaneous Melanoma: The Future Role of Circulating Melanoma Cells in Prognosis and Management,” Clin Exp Metastasis 28(4):327e36 (2011)). This cell must also have the ability to self-renew and differentiate to drive continuous heterogeneous tumor growth. Cell sub-populations within the primary tumor that have the ability to self-renew, differentiate, and show increased in vivo tumorigenicity are called cancer stem cells (CSCs) (Schatton and Frank, “Cancer Stem Cells and Human Malignant Melanoma,” Pigment Cell Melanoma Res 21(1):39e55 (2008)). Identifying CSCs and characterizing their cellular origin, phenotype and the mechanisms that confer their tumor-initiating properties have important ramifications for understanding cancer biology and, ultimately, developing cancer cures.
Melanomas are invasive, heterogeneous tumors that are highly resistant to conventional therapies (Schatton and Frank, “Cancer Stem Cells and Human Malignant Melanoma,” Pigment Cell Melanoma Res 21(1):39e55 (2008); Fang et al., “A Tumorigenic Subpopulation with Stem Cell Properties in Melanomas,” Cancer Res 65(20):9328e37 (2005); Schatton et al., “Identification of Cells Initiating Human Melanomas,” Nature 451(7176):345e9 (2008); Boiko et al., “Human Melanoma-initiating Cells Express Neural Crest Nerve Growth Factor Receptor CD271,” Nature 466(7302):133e7 (2010); Schmidt et al., “Eradication of Melanomas by Targeted Elimination of a Minor Subset of Tumor Cells,” Proc Natl Acad Sci USA 108(6):2474e9 (2011); Yang and Chapman, “The History and Future of Chemotherapy for Melanoma,” Hematol Oncol Clin North Am 23(3):583e97 (2009)). The ability of melanoma to relapse after treatment suggests a lack of knowledge about the biological properties and phenotypes of constituent cell sub-populations and the ineffectiveness of current therapies to eradicate the tumor-initiating cells (DiFronzo et al., “Increased Incidence of Second Primary Melanoma in Patients with a Previous Cutaneous Melanoma,” Ann Surg Oncol 6(7):705e11 (1999); Cummins et al., “Cutaneous Malignant Melanoma,” Mayo Clin Proc 81(4):500e7 (2006)). Studies have correlated the tendency of melanoma cells to propagate in vitro as non-adherent spheroids with increased tumor cell invasiveness (Fang et al., “A Tumorigenic Subpopulation with Stem Cell Properties in Melanomas,” Cancer Res 65(20):9328e37 (2005); Bates et al., “Spheroids and Cell Survival,” Crit. Rev Oncol Hematol 36(2e3):61e74 (2000); Sodek et al., “Compact Spheroid Formation by Ovarian Cancer Cells Is Associated with Contractile Behavior and an Invasive Phenotype,” Int J Cancer 124(9):2060e70 (2009); Monzani et al., “Melanoma Contains CD133 and ABCG2 Positive Cells with Enhanced Tumourigenic Potential,” Eur J Cancer 43(5):935e46 (2007)), and resistance to chemotherapeutics (Bates et al., “Spheroids and Cell Survival,” Crit. Rev Oncol Hematol 36(2e3):61e74 (2000); Mueller-Klieser, “Three-dimensional Cell Cultures: From Molecular Mechanisms to Clinical Applications,” Am J Physiol 273(4 Pt 1):C1109e23 (1997); Minchinton and Tannock, “Drug Penetration in Solid Tumours,” Nat Rev Cancer 6(8):583e92 (2006)). This has lead to the increasing use of the in vitro sphere assay to study melanoma and other types of CSCs (Pastrana et al., “Eyes Wide Open: A Critical Review of Sphere-Formation as an Assay for Stem Cells,” Cell Stem Cell 8(5):486-98 (2011); Held et al., “Characterization of Melanoma Cells Capable of Propagating Tumors from a Single Cell,” Cancer Res 70(1):388e97 (2010); Fang et al., “A Tumorigenic Subpopulation with Stem Cell Properties in Melanomas,” Cancer Res 65(20):9328e37 (2005); Minchinton and Tannock, “Drug Penetration in Solid Tumours,” Nat Rev Cancer 6(8):583e92 (2006); Perego et al., “Spheres of Influence in Cancer Stem Cell Biology,” J Invest Dermatol 131(2):546e7 (2011)). The first evidence that the spheroid cell culture could enrich for CSCs in melanoma came in 2005, when cells propagating as melanospheres exhibited the capacity to undergo multi-lineage differentiation and they exhibited a 10-fold increase in tumorigenicity (ability to form tumors in mice) when compared to cells propagating as a monolayer (Fang et al., “A Tumorigenic Subpopulation with Stem Cell Properties in Melanomas,” Cancer Res 65(20):9328e37 (2005)). Ramgolam et al. (Ramgolam et al., “Melanoma Spheroids Grown under Neural Crest Cell Conditions Are Highly Plastic Migratory/invasive Tumor Cells Endowed with Immunomodulator Function,” PLoS ONE 6(4):e18784 (2011)) positively correlated melanoma cells growing as spheroids with a more aggressive phenotype; exhibiting enhanced migratory/invasive characteristics, immune evasion capacity, and ability to differentiate along mesenchymal lineages. They did not, however, observe enhanced self-renewal or tumor initiating capacity in xenotransplantation experiments. Perego et al. (Perego et al., “Spheres of Influence in Cancer Stem Cell Biology,” J Invest Dermatol 131(2):546e7 (2011); Perego et al., “Heterogeneous Phenotype of Human Melanoma Cells with in vitro and in vivo Features of Tumor-initiating Cells,” J Invest Dermatol 130(7):1877e86 (2010)) reported xenotransplantion of melanosphere cells into immunocomprised mice resulted in larger tumors compared to recipients of adherent melanoma cells; however, no significant difference in tumorigenicity was observed. The functional ambiguity of cells propagating as melanospheres and the inability to associate a unique set of stem cell surface markers with tumor initiating capacity (Quintana et al., “Efficient Tumour Formation by Single Human Melanoma Cells,” Nature 456(7222):593e8 (2008); Quintana et al., “Phenotypic Heterogeneity Among Tumorigenic Melanoma Cells from Patients that Is Reversible and not Hierarchically Organized,” Cancer Cell 18:510e23 (2010)) has led some to challenge the usefulness of the melanosphere cell culture (Schatton and Frank, “The in vitro Spheroid Melanoma Cell Culture Assay: Cues on Tumor Initiation?” J Invest Dermatol 130(7):1769e71 (2010)). It is plausible, however, that these discrepancies may reflect differences in the various methods used to propagate melanospheres and limitations with the methods used to characterize the constituent cell sub-populations (Kubick and Roop, “A Fitness Model for Melanoma-initiating Cells,” Pigment Cell Melanoma Res 24:396e400 (2011)).
Common approaches used to propagate spheroids include culturing cells in highly mitogenic stem cell media (Fang et al., “A Tumorigenic Subpopulation with Stem Cell Properties in Melanomas,” Cancer Res 65(20):9328e37 (2005); Ramgolam et al., “Melanoma Spheroids Grown under Neural Crest Cell Conditions Are Highly Plastic Migratory/invasive Tumor Cells Endowed with Immunomodulator Function,” PLoS ONE 6(4):e18784 (2011)), on soft agar (Hamburger and Salmon, “Primary Bioassay of Human Tumor Stem Cells,” Science 197(4302):461e3 (1977)), or on Matrigel (Monzani et al., “Melanoma Contains CD133 and ABCG2 Positive Cells with Enhanced Tumourigenic Potential,” Eur J Cancer 43(5):935e46 (2007); Schatton and Frank, “The in vitro Spheroid Melanoma Cell Culture Assay: Cues on Tumor Initiation?” J Invest Dermatol 130(7):1769e71 (2010); Yeung et al., “Cancer Stem Cells from Colorectal Cancer-derived Cell Lines,” Proc Natl Acad Sci USA 107(8):3722e7 (2010); Civenni et al., “Human CD271-positive Melanoma Stem Cells Associated with Metastasis Establish Tumor Heterogeneity and Long-term Growth,” Cancer Res 71(8):3098e109 (2011)). These methods are complex, and expensive as growth is usually done under limiting dilution conditions where cells are plating at low density (≦1 cell per well). This is a time consuming process (3-4 weeks), that often requires special media that must be developed through tedious trial-and-error processes. Cells are often prospectively sorted by expression of stem cell markers using flow cytometry, which is an invasive process that can alter cell function and/or viability. In vivo tumorigenicity is characterized using cumbersome and time consuming (1-3 months) animal models that vary in permissiveness; often using serial dilution to quantify the frequency or percent of tumor initiating cells within the heterogeneous cell sample (Fang et al., “A Tumorigenic Subpopulation with Stem Cell Properties in Melanomas,” Cancer Res 65(20):9328e37 (2005); Schatton et al., “Identification of Cells Initiating Human Melanomas,” Nature 451(7176):345e9 (2008); Monzani et al., “Melanoma Contains CD133 and ABCG2 Positive Cells with Enhanced Tumourigenic Potential,” Eur J Cancer 43(5):935e46 (2007); Quintana et al., “Efficient Tumour Formation by Single Human Melanoma Cells,” Nature 456(7222):593e8 (2008); Quintana et al., “Phenotypic Heterogeneity Among Tumorigenic Melanoma Cells from Patients that Is Reversible and not Hierarchically Organized,” Cancer Cell 18:510e23 (2010); Welte et al., “Cancer Stem Cells in Solid Tumors: Elusive or Illusive?” Cell Commun Signal 8(1):6 (2010)). In vitro clonogenic potential may be determined using limiting dilution cell culture to quantify the frequency of single cells that grow into clonal pure spheres of ˜50 cells (Singh et al., “Identification of a Cancer Stem Cell in Human Brain Tumors,” Cancer Res 63(18):5821e8 (2003)). Given the above mentioned experimental limitations, there is an unmet need for rapid, cost effective nonanimal methods to enrich CSCs from heterogeneous cell sample and to determine the clonogenic potential of constituent cells in a manner that can predict tumorigenic potential.
The present invention is directed to overcoming these and other deficiencies in the art.